衣康酸(itaconic acid)為一種不飽和雙碳有機酸，因此具有將單體聚合成聚合體的特性，故可應用於合成樹脂、纖維、塑料及橡膠的原料，廣泛應用於化學、高分子工業上。此研究將利用微生物產生纖維水解酵素水解農業纖維廢棄物，並使用pH應答型高分子包埋後利用共同糖化發酵產衣康酸。過去研究無法利用較廉價的基質，導致生產衣康酸(itaconic acid)等化學品失去競爭力。雖然嘗試透過基因工程菌株達到利用纖維素之目的。但是酵素生產與產物生成往往無法兩全。因此，本計畫將提出一項可能的解決方法。首先提高pH，生產酵素達到醣化目的，再降低pH 進行衣康酸生產之目的。此研究預設為2 年，即利用pH 應答型材質絮凝固定化Bacillus sp 來探討以農業纖維素為基質行兩階段pH控制於衣康酸產量的影響。第一年:培養Bacillus sp生產纖維水解酵素後以pH應答型材質進行絮凝吸附固定化: a. 微生物產纖維水解酵素:一般所說纖維素水解酵素(cellulase)為作用於纖維素的酵素總稱，能將纖維素水解成可溶性醣類，供給微生物生長代謝所需。具纖維素分解能力的微生物，往往其生長條件類似化學品生產條件，因此產生爭奪基質的問題，透過包埋與pH調控可以改變此狀況。 b. pH應答型聚合物:環境敏感型高分子聚合物主要應用於生醫及藥物控制釋放，而 pH敏感型材料能夠依賴pH值的變化，表現出膨脹與收縮的形變；本實驗操作的海藻酸為pH敏感型材質，於鹼性溶液中親水性增加；酸性溶液中親水性降低，故將此特性延伸於菌體固定化的應用，將有助於改善傳統包埋技術的缺點。第二年:以纖維廢棄物為基質，藉Aspergillus terreus.生產衣康酸(itaconic acid): a. 農業纖維廢棄物: 台灣目前水稻每年約產生150萬噸廢棄稻稈，長期大都以焚燒方式處理，造成空氣污染等問題。若可利用發酵程序生產衣康酸，不但能兼具能源與環保雙重價值，以操作與成本考量，也相當具有競爭力。一般來說，改變環境因子及操作手法都將有利於促進產物提升。 b. 共同糖化發酵法:就以往的SSF 發酵而言，纖維水解通常以基改菌種來進行共同糖化，不過仍存在菌種特性不穩定、高研究成本等缺點；而利用環境應答型材料進行包埋，再透過改變環境因素，考量於酵素活性與菌體生長兩者適性的切換，以便行連續式或饋料式操作。本研究之創新在於以下敘述: 1. 首度探討in-situ絮凝固定化對於菌體生長於不同pH值的環境下，酵素產物與活性的影響。 2. 利用pH應答型材料作為菌體包埋與酵素釋放的研究，以期建立共生培養系統，致使能連續式操作降低成本。 3. 作纖維廢棄物為基質，進而調控pH值來進行共生與共同糖化發酵的研討，來達到目標產物之發酵。相信本研究成果不僅具學術性，也具商業可行性，對學術與相關產業有重要影響。 Agricultural waste is the largest resource of cellulose which can be potential substrates for production of valuable chemicals such as itaconic acid. Itaconic acid is the monomer of many commercial resins, fabrics, plastics and rubbers. Unfortunately, it is still not economic competitive if glucose is used as the substrate. In general, the cost of enzymes is relatively high; thus, the production of enzyme is the most economical approach to obtain the required cellulase. However, seperation of the enzyme become the major barrier of this approach. Co-culture is another alternative using the enzyme from fermentation; however, competition of nutrients between the two culture become the challenge of this approach. Metabolic engineering of the culture to produce both enzyme and itaconic acid is one of the most exciting approach; however, the efficiency of itaconic acid production is compromised. We also find that the fermentation conditions for enzyme production by Bacillus are obviously different from those of itaconic acid production by Aspergillus. Thus, the main objectives of this proposal are to develop in-situ immobilization of cellulase producing cells by pH-responsive hydrogel, and to develop two-stage pH-control strategy for converting agricultural waste into itaconic acid. And, it might take two-year efforts to reach the goals. In other words, the project is 2-year proposal. 1st year: The objective of this year is to develop in-situ immobilization of cellulase producing cells by pH-responsive hydrogel. Bacillus could produce cellulase under a high pH range, and alginate as pH-responsive gel could be use as the material for in-situ immobilization to separate cell from broth in a low pH range. 2nd year: The objective of this year is to convert agricultural waste into itaconic acid by a two-stage pH control strategy using a co-culture and in-situ immobilization system. The novelty of this proposal is described as below: 1. The project would be the first using in-situ immobilization system to produce required cellulase to convert agricultural wastes. 2. The project would be the first to manipulate pH-responsive alginate to immobilize cells and to release cells under different culture pH. 3. The project would be the first approach to utilize cellulose by SSF process without using metabolic approach or any other cellulose pretreatment. We strongly believe that the results of this project would be beneficial to both academia and industry. 研究期間：10008 ~ 10107
